Cells interact with biochemically and structurally distinct forms of extracellular matrix in different tissues, at different stages of embryonic development, and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. What unique mechanisms do mammalian cells use to migrate through different three-dimensional (3D) extracellular matrix environments compared to flat cell culture substrates? 2. What distinct signal transduction mechanisms and modes of cell movement control cell behavior in different 3D microenvironments? We are exploring which elements of the classical models of cell motility and signaling established using regular 2D cell culture are valid in the structurally complex 3D environments found in tissues. We have identified a variety of differences in cell migration and signaling between 2D and 3D environments, but also between different 3D environments, such as collagen-based matrices that differ in architecture compared to fibronectin-rich, crosslinked, cell-derived matrices. In these matrices, primary human dermal fibroblasts migrate at different speeds and with distinct modes of migration. Reliable, reproducible characterization of cell interactions with adjacent collagen fibrils requires careful attention to methods of collagen labeling by fluorophores, polymerization into fibrillar meshworks, and analysis by confocal microscopy, for which detailed methods were recently published. Human fibroblastic cells in 3D collagen environments, regardless of their local stiffness and micro-architecture, were previously shown to display markedly elevated levels of integrin activation, clustering, and cell adhesion formation compared to cells cultured on flat collagen matrices in cell culture. This enhanced integrin activation was accompanied by a requirement for cellular contractility, apparently in order for these cells to be able to detach effectively from their enhanced integrin-mediated attachments to 3D fibrils to permit efficient cell migration. The next phase of this study is characterizing the myosin II-dependent mechanisms of human fibroblast migration in 3D collagen microenvironments, whereas myosin II is not required when these same cells migrate on 2D surfaces. In addition, we are comparing the alterations in mechanisms of migration after malignant transformation by an in-depth characterization of the modes of 3D cell migration used by human tumor cells. We are tracking local movements of cellular features of migrating cells that include cell-matrix adhesions, the leading edge, nucleus, and trailing edge of cells compared to the local adjacent collagenous matrix. These approaches are identifying distinct modes of 3D cell migration dependent on localized sites and dynamics of extracellular matrix deformation using non-muscle myosin IIA versus myosin IIB.